Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

The mature human gut microbiota is established during the first years of life, and altered intestinal microbiomes have been associated with several human health disorders. Escherichia coli usually represents less than 1% of the human intestinal microbiome, whereas in cystic fibrosis (CF), greater than 50% relative abundance is common and correlates with intestinal inflammation and fecal fat malabsorption. Despite the proliferation of E. coli and other Proteobacteria in conditions involving chronic gastrointestinal tract inflammation, little is known about adaptation of specific characteristics associated with microbiota clonal expansion. We show that E. coli isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. E. coli isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with E. coli from healthy controls, and unrelated CF E. coli strains have independently acquired this growth trait. Furthermore, CF and control E. coli isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth-promoting transcriptional profile, control isolates engage stress and stationary-phase programs, which likely results in slower growth rates. Our results indicate that there is selection of unique characteristics within the microbiome of individuals with CF, which could contribute to individual disease outcomes.

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.

As mediators of adhesion, autoaggregation and bacteria-induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili-dependent functions require different ranges of pili numbers.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-β-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Les pili de type IV (PT4), certainement les organelles les plus repandues des bacteries a Gram-negatif, sont des machineries a multiples fonctions qui jouent un role crucial dans la pathogenese de nombreux pathogenes humains, notamment notre modele Neisseria meningitidis. L’assemblage des PT4 necessite une machinerie complexe incluant au moins vingt proteines localisees dans la membrane interne, le periplasme et la membrane externe. Certaines de ces proteines ne sont pas necessaires pour la biosynthese des PT4, mais supportent les fonctions qui leur sont associees. Ces proteines, appelees pilines mineures, sont au nombre de trois. Par l’analyse phenotypique des mutants dans les genes codant pour les pilines mineures, le role de chacune a pu etre determinee. Ainsi la piline mineure ComP est necessaire pour la competence pour la transformation d’ADN, PilV est requise pour la deformation de la membrane plasmique de la cellule hote et PilX est essentielle pour l’adhesion des bacteries sur les cellules epitheliales et endotheliales, la formation d’agregats bacteriens et la deformation de la membrane plasmique de la cellule hote. De nombreuses similarites avec la piline majoritaire laissent penser que les pilines mineures s’inserent dans la fibre des PT4 pour exercer leurs fonctions, bien que ceci n’a jamais ete demontre. Si on connait bien les fonctions des pilines mineures, leur mode d’action n’est toujours pas compris. L’objectif global de ce travail de these a ete de comprendre comment une fibre proteique peut assurer une diversite de fonctions aussi importante. Pour y parvenir, l’etude du mode d’action des pilines mineures a ete entreprise. Contrairement a ce qui prevalait dans le modele dominant, les pilines mineures PilV et PilX exercent leur fonction a partir de l’espace periplasmique pour moduler la quantite de pili exprimes en surface. En effet, les mutants pilV et pilX presentent respectivement des defauts de piliation de l’ordre de 39% et de 63% par rapport a la souche sauvage. Ces defauts expliquent cependant les phenotypes des mutants. En effet, l’ensemble des fonctions dependantes des PT4 necessite une forte quantite de PT4, soit au moins 40% pour l’agregation et l’adhesion et 70% pour le declenchement de la reponse cellulaire. Ces resultats revelent que les pilines mineures sont impliquees dans la biogenese des PT4 plutot que dans le support biochimique direct de leurs proprietes. Le defaut de piliation de ces mutants est restaure par l’absence de retraction, indiquant que les pilines mineures PilV et PilX jouent un role dans la stabilite des PT4. Nous avons egalement montre que la piline mineure ComP est necessaire pour la piliation et qu’elle presente une fonction redondante avec la piline mineure PilV. Afin de comprendre comment les pilines mineures PilV et PilX exercent leur role sur la quantite de pili exprimes en surface, nous avons realise une etude structure/fonction de ces deux proteines. Nous avons observe une absence de piliation, en bloquant les pilines PilV et PilX dans la membrane interne, indiquant une interaction directe avec la machinerie des PT4 probablement via la piline majeure PilE. Nous avons egalement montre qu’il existe une interaction entre les pilines mineures et PilE au niveau de la membrane interne et en amont de l’assemblage des pili. Ces resultats, obtenus par une technique de pontage disulfure, ont cependant besoin d’etre confirmes par des controles supplementaires. Par une strategie de mutagenese, nous avons enfin mis en evidence que la region D de PilV et les boucles α/β et β2/β3 de PilX sont necessaires a leur fonctionnement. Ces travaux ont permis de montrer que la quantite de pili exprimes par la bacterie est un facteur determinant pour definir les proprietes des PT4. Les pilines mineures agissent au niveau du periplasme pour promouvoir la biosynthese des pili, ce qui met en avant le role direct de la piline majeure PilE dans les fonctions associees aux PT4.

The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.